The presence of the enzyme Na+K+ATPase in the bovine adrenal gland has been studied using 3 different approaches: 3H-ouabain binding, catecholamine (CA) secretion evoked by cardiac glycosides and 86Rb uptake sensitive to cardiac glycosides. We found only one homogeneous population of 3H-ouabain binding sites likely located on the plasmatic membrane of chromaffin cells with a Kd=13.6 plus minus 1.2nM and a Bmax=3.15 plus minus 0.21pmol/mg protein. The 3H-ouabain binding was specific and could be displaced by other cardiac glycosides with an order of potency of ouabain more than acetyldigitoxin more than digitoxin. Blockade of Na+K+ATPase with cardiac glycosides induced CA release from isolated bovine chromaffin cells. This CA release was both Ca2+ and temperature dependent. The order of potency for cardiac glycosides was ouabain more than strophanthidin more than digitoxin. We also tried to correlate the CA release induced by cardiac glycosides to the Na+K+ATPase activity measured as 86Rb or 42K+ uptake in isolated chromaffin cells. 86Rb uptake reached a plateau at 60 minutes. A similar course was obtained with 42K+. 86Rb+ uptake was blocked in a dose-dependent manner by cardiac glycosides, being ouabain the most potent drug.